Outcome-Related Signatures Identified by Whole Transcriptome Sequencing of Resectable Stage III/IV Melanoma Evaluated after Starting Hu14.18-IL2

Abstract

Purpose: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence.

Experimental design: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing.

Results: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors.

Conclusions: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy.

Trial registration: ClinicalTrials.gov NCT00590824.

Conflict of interest statement

Conflict of interest statement: The authors report no financial or other conflicts of interest to disclose related to this publication.

©2020 American Association for Cancer Research.

Figures

Figure 1.. Morphologic Tumor Infiltrating Lymphocytes (TILs) Assessment and the Stromal Gene Signature Prognosticate Recurrence Free and Overall Survival after hu14.18-IL2 Immunocytokine (IC) Treatment, but not Before IC Treatment.

(A-C) Kaplan Meier curves of patients stratified by TIL assessment are shown. Panel A demonstrates stratification by median TIL assessment is prognostic for recurrence free survival (RFS) and overall survival (OS) within all patients. Patients with below the median TIL levels demonstrate worse outcomes. Panels B and C show this prognostic effect on RFS and OS, respectively, retained in treatment Group A, but not in treatment Group B. (D-F) Kaplan Meier curves of patients stratified by the stromal signature (37) are shown. Panel D demonstrates that the stromal signature is also prognostic of RFS and OS within all patients. Patients with below the median Stromal Signature demonstrate worse outcomes. Similarly, panels Eand F show this prognostic effect on RFS and OS, respectively, retained in treatment Group A, but not in treatment Group B.

Figure 2.. Gene Signatures Prognosticate Favorable Patient Survival in hu14.18-IL2 Treated Tumors, but Not in Group-B (Untreated) Tumors

Tree plots of the 25 immunologic signatures consistently demonstrated favorable hazard ratios for RFS and OS. (A) For Group A patients, ten (40%) of these signatures showed statistically significant favorable hazard ratios (HRs) of RFS (and ten others showed a trend, 0.01< p ≤ 0.1) but none were significant (or showed a trend) for Group B patients. (B) For Group A patients six (24%) of these signatures showed statistically significantly favorable HRs of OS (and eight others showed a trend, 0.01< p ≤ 0.1), but none were significant (or showed a trend) for Group B patients. Hazard ratios are –Ln (natural log) transformed such that a point estimate of 1.0 denotes a HR of 0.368 and a point estimate of −1.0 denotes a HR of 2.72, thus points plotted to the right of 0.0 indicate favorable outcome and those to the left indicate unfavorable outcome. Legend: *** = p-value <0.001; ** = p-value <0.01, * = p-value <0.05, # = p-value <0.1. The p-values are from the likelihood ratio test (it performs better when sample size is small), whereas the 95% confidence intervals are based on the Wald test.

Figure 3.. Granular Immunologic Gene Expression Demonstrates a Predominance of Parameters with Favorable Prognostic Value Rather than Unfavorable Prognostic Value.

Volcano plots of granular immunologic gene expression are plotted with hazard ratios against significance levels (A-D). Panels A and C represent the Group A patients’ (n=13) RFS and OS relationships, respectively, against these immunologic genes.Panels B and D represent the Group B patients’ (n=8) RFS and OS relationships, respectively, against these immunologic genes. Several genes show significantly favorable hazard ratios (red points) in Panels A and C. Among these CCR3 (a chemokine receptor on eosinophils and helper T cells) and VNN1 (a membrane protein felt to influence hematopoietic and T cell trafficking, both labelled in red) stood out as highly significant, and associated with a very skewed HR, prognostic of favorable RFS and OS in Group A. The horizontal dotted line represents a p-value of 0.01. The vertical dotted line represents a hazard ratio of 1.0.

Figure 4.. Expression of Melanoma Markers and Oncogenes Portends Unfavorable RFS and OS in Group A but not Groups B. Expression of Apoptotic Marker Genes Portends Favorable RFS and OS in Group A but not Group B.

Gene expression level relationships between melanoma related (top) and apoptosis related (bottom) expression and RFS and OS are shown in heat map form. Melanoma related genes were selected from molecular markers routinely used in diagnostic anatomic pathology. The green to red legend demonstrates either favorable prognosis (HR <1, green) or unfavorable prognosis (HR >1, red), respectively. The blue coloring represents level of significance starting with light blue at p=0.10 and becoming deeper blue with increasing significance. These melanoma and apoptotic parameters tend to prognosticate unfavorable or favorable survival, respectively, in Group A more than they do in Group B.

Figure 5.. IC Treatment reveals a Predominance of Increased Rather than Deceased Gene Expression.

(A) Gene expression for individual genes are plotted with Log 2 fold changes from naïve [8 in Group B and 2 untreated (No IC)] patients to 13 Group A hu14.18-IL2 treated patients (y-axis) against significance levels (x-axis). There is a marked preponderance of genes that are significantly increased (upper right quadrant), indicating an overall increase in gene expression of transcripts after treatment with hu14.18-IL2. (B) Gene expression plot for individual cancer-related genes (annotated from the Human Protein Atlas) with Log 2 fold changes from 10 naïve patients to 13 Group A patients demonstrates a preponderance of significantly increased (upper right quadrant), but not decreased, expression of genes in Group A vs. naïve tumors. (C) Gene expression plot for individual ssGSEA CIBERSORT immune genes with Log 2 fold changes from 10 naïve patients to 13 Group-A patients demonstrates a preponderance of significantly increased, but not decreased, expression of genes in Group A vs. naïve tumors. There are 52 genes (at p<0.01) and 610 genes (at p<0.05) that show significant fold difference between treated and hu14.18-IL2 naïve tumors. The vast majority of these were upregulated rather than downregulated. Fold change is Log2 transformed such that a point estimate of 1.0 is denotes a fold change of 2.0 and a point estimate of −1.0 denotes a fold change of 0.5. In A, B, and C, the vertical dotted lines represent p-values of 0.05 and 0.01. The horizontal dotted line represents a ratio of 1.0 for expression of the individual gene in the tumors from 13 Group A patients vs. in the 10 naïve tumors. Immunologic genes were taken from all 645 immunologically related genes that are included in our ssGSEA analysis. For Fig. 5A, 5B and 5C, to the right of the dot plots for gene expression ratio vs. p value are box plots and density histograms of those points that are significant (p<0.01), together with those that have 0.01< p < 0.05.

Figure 6.. Pathway -Level Enrichment Analysis of Gene Sets Whose Expression Correlates with Survival.

The pathway-level enrichment analysis indicates that genes involved in innate immune response are significantly over-represented in patients with improved OS (HR green top left panels), but not in the untreated tumors (n = 8, Group B). A complete list of 53 pathways significant associated with favorable OS in Group A patients may be found in Supplemental Figure S15. In contrast, only a few pathways including ectodermal differentiation and activation of SMO were found to associate with Group A patients with decreased OS (pink top right panel). Pathways associated with improved RFS (HR < 1) in Group A patients include chemokine and cytokine signaling (green middle left panels). In contrast, pathways associated with unfavorable RFS (HR > 1) in Group A patients include glycolysis and other glucose metabolism (pink middle right panels). In Group B patients, pathways associated with focal adhesion associated with favorable OS in Group B (green bottom left panel). In contrast, genes involved in de-ubiquitination and processing of ubiquitinated proteins are over-represented in patients with unfavorable OS (HR > 1) in Group B (pink bottom right panels). Pathways are ranked by their log (1/q-value) values and colored blue when the q-value <0.05 with darkening blue representing increasing statistical significance. The q-value is the false discovery rate (FDR) adjusted p-value and provides a correction for multiple tests (that is, takes into account the total number of pathways used in the analysis). The q-value is commonly used in genome-wide studies.